Mutations in the type I ryanodine receptor (RyR1) are associated with a variety of human muscle diseases including malignant hyperthermia (MH), MH with cores, central core disease (CCD), multi-minicore disease, and others. RyR1-related myopathies are among the most common group of non-dystrophic muscle diseases and are associated with significant clinical disabilities, often including wheelchair dependence, severe scoliosis, respiratory failure, and can result in premature death in childhood. Currently there are no therapies for these devastating myopathies. MH mutations in RyR1 are frequently associated with heat stress, and/or exercise-induced heat stroke and/or rhabdomyolysis. CCD is a congenital myopathy associated with metabolically inactive central cores in skeletal muscle fibers, but the presence of cores is highly variable (despite the name) and the severity of the disease does not correlate with cores. We have shown that some CCD mutations increase Ca2+ leak from the sarcoplasmic reticulum, while others decrease Ca2+ permeation through RyR1. These finding raise the question of how opposing functional effects on RyR1 can result in related diseases. To answer this central, unresolved question and aid in the development of new interventions, we created mouse models of MH with cores (Y524S, YS) and CCD (I4895T, IT). Heterozygous YS mice are susceptible to anesthetic and heat-induced sudden death and also exhibit an age-dependent myopathy characterized by mitochondrial damage and the formation of amorphous cores. Heterozygous IT mice are not heat sensitive, but display decreased exercise capacity, deceased muscle fiber cross-sectional area, and a myopathy that increases with age. In this renewal application, we propose to define the cellular and molecular mechanisms by which functionally opposing RyR1 mutations produce these distinct phenotypes and then use these findings to develop new, mechanism-based therapeutic interventions for MH and CCD. Our working hypothesis is that the YS mutation drives the disease via increased RyR1 Ca2+ leak, activation of the mitochondrial permeability transition pore (mPTP), and oxidative stress, while the IT mutation enhances SR Ca2+ content, which leads to increased ER stress, p53 expression, mitochondrial damage and apoptosis. To test this hypothesis, we propose to: 1) Define the role of altered cytosolic, mitochondrial, and SR lumenal Ca2+ signaling and mPTP activation in the YS and IT myopathies. 2) Define the roles of RyR1 post- translational modifications. 3) Define the role of p53 in the IT myopathy and the enhanced heat sensitivity of YS mice. 4) Assess the potential of S107, NAC, 4PBA, and pifithrin- as interventions for MH and CCD. There are currently no therapies to treat RyR1-related myopathies. This multi-PI application is designed to address this need by carefully delineating specific cellular pathways that underlie disease processes and then to use this information to develop and test several novel therapeutic interventions with distinct mechanisms of action.